Cell counting and sample chamber and methods of fabrication

ABSTRACT

The invention generally relates to analytical and monitoring systems useful for analyzing and measuring cells and biological sample. More particularly, the invention relates to a unique cell counting chamber, e.g., a thin gap fluidic cell chamber for both bright field and fluorescent imaging of bacteria or parasites, and methods for making the same.

PRIORITY CLAIMS AND RELATED APPLICATIONS

This application claims the benefit of priority from U.S. patentapplication Ser. No. 13/519,283, filed Jan. 3, 2013, which is the U.S.national phase of and claims the benefit of priority fromPCT/US11/21676, filed Jan. 19, 2011, which claims the benefit ofpriority from U.S. Provisional Application Ser. No. 61/296,778, filedJan. 20, 2010, the entire content of each of which is incorporatedherein by reference in its entirety.

TECHNICAL FIELDS OF THE INVENTION

The invention generally relates to analytical and monitoring systemsuseful for analyzing and measuring cells and biological sample. Moreparticularly, the invention relates to a unique sample chamber, e.g., athin gap fluidic cell counting chamber useful for both bright field andfluorescent imaging of bacteria or parasites, and methods for making andusing the same.

BACKGROUND OF THE INVENTION

Detection, identification, quantification, and characterization ofbiomolecules or cells of interest through testing of biological samplesis an important aspect in the fields of medical diagnostics andbiomedical research. Biological solutions, such as blood, spinal fluid,cell culture and urine, are routinely analyzed for their microscopicparticle concentrations.

Existing bacteria cell counting methods include inexpensive but manualcell counting hemacytometer and expensive large instruments such as flowcytometer, plate reader, as well as bacterial colony measurement. Themanual cell counting device is extremely tedious and inconsistent and isprone to human errors and cleaning procedures. Flow cytometry requireslarge quantity of samples, due to the utilization of lasers, is veryexpensive and requires well-trained professionals to perform theinstrument in order to obtain accurate results. Bacteria colony andmeasurement by optical density has also been used in cell counting, butthe preparation is tedious and prone to contamination. In addition, itrequires days and even weeks for the bacteria culture to be accuratelymeasurable.

Recently, an automated cell counting instrument has been developed byNexcelom Bioscience that utilizes a microfluidic counting chamber, whichcan automatically count cells larger than 2 μm in diameter.

There is, however, a pressing need for efficient and consistent imagingand counting instrument and methods for biological particles withdiameters less than 2 μm, such as bacteria or parasites.

SUMMARY OF THE INVENTION

The invention is based, in part, on the discovery of a novel thin gapfluidic chamber design and efficient and cost-effective methods offabrication and manufacture. In order to consistently image and countthe particles, a thin gap fluidic sample chamber is required to minimizethe effect of multiple imagine planes. Depending on the particles sizes,the novel fabrication method can be used to create the appropriatechamber gap height. The cell chamber of the invention can be used forboth bright field and fluorescent imaging of bacteria, parasites,protein aggregates, or larger mammalian, insect, or plant cell types.

In one aspect, the invention generally relates to a sample chambersuitable for holding a liquid sample for optical imaging. The samplechamber includes: a top layer; an adhesive layer; a bottom layer; asample holding cavity between the adhesive layer and the bottom layerhaving a fixed depth of from about 0.5 μm to about 1,000 μm; and anassess port for placing a sample into or out of the chamber; wherein atleast a portion of the adhesive layer is optically clear in the range ofabout 250 nm to about 10,000 nm in wavelength, and wherein at least aportion of the top layer and the bottom layer is optically clear in therange of about 300 nm and about 10,000 nm in wavelength.

The bottom layer of the sample chamber further includes a base film.

In certain embodiments, the sample holding cavity of the sample chamberhas a fixed depth of from about 1 μm to about 100 μm. In certainpreferred embodiments, the sample holding cavity has a fixed depth offrom about 2 μm to about 50 μm. In certain preferred embodiments, thesample holding cavity has a fixed depth of from about 2 μm to about 20μm. In certain other preferred embodiments, the sample holding cavityhas a fixed depth less than about 2 μm. In certain other preferredembodiments, the sample holding cavity has a fixed depth less than about1 μm.

In some embodiments, at least a portion of the adhesive layer isoptically clear in the range of about 300 nm to about 10,000 nm inwavelength, and wherein at least a portion of the top and the bottomlayers is optically clear in the range of about 300 nm and about 10,000nm in wavelength.

The top layer of the sample chamber may be made of a material selectedfrom the group consisting of polyester, polycarbonate, poly(methylmethacrylate) (or PMMA), polystyrene and cyclic olefin copolymer, andother optically clear plastic films.

The adhesive layer of the sample chamber may be a pressure-sensitiveadhesive layer that includes a material selected from acrylic oracrylate adhesive.

The bottom layer may include an irradiation or thermally cured material.For example, the bottom layer may be a UV-cured polymeric material. Incertain preferred embodiments, the UV-cured materials are selected fromthe group consisting of UV-cured acrylate, urethane and epoxy.

In certain preferred embodiments, the sample chamber further includes abacking layer that is bond to the bottom layer. The backing layer mayinclude a material selected from the group consisting of polyester,polycarbonate, PMMA, polystyrene and cyclic olefin copolymer, and otheroptically clear plastic films.

In another aspect, the invention generally relates to a sample chambersuitable for holding a biological sample for optical imaging. The samplechamber includes: an optically clear bottom layer; an optically cleartop layer; an optically clear adhesive layer binding the top layer onone side and forming a sample holding chamber on the other side with anirradiation- or thermally-cured polymer layer, wherein the sampleholding chamber having a fixed depth from the adhesive layer to theirradiation- or thermally-cured polymer layer of from about 0.5 μm toabout 1,000 μm; an inlet port for introducing a sample into the chamber;and an outlet port for removing a sample from the chamber (the sampleholding cavity).

In some embodiments, the sample holding chamber has a fixed depth offrom about 1 μm to about 100 μm. In certain preferred embodiments, thesample holding chamber has a fixed depth of from about 2 μm to about 50μm. In certain preferred embodiments, the sample holding chamber has afixed depth of from about 2 μm to about 20 μm. In certain otherpreferred embodiments, the sample holding chamber has a fixed depth ofless than about 2 μm. In certain other preferred embodiments, the sampleholding chamber has a fixed depth of less than about 1 μm.

For some applications, it is preferred that at least a portion of theadhesive layer is optically clear in the range of about 250 nm to about10,000 nm in wavelength.

The top layer may be made of a material selected from the groupconsisting of polyester, polycarbonate, PMMA, polystyrene and cyclicolefin copolymer.

The adhesive layer may be a pressure-sensitive adhesive layer comprisinga material selected from acrylic or acrylate adhesive.

The bottom layer may include an irradiation or thermally cured material.In certain preferred embodiments, the bottom layer comprises a UV-curedpolymeric material, for example, a material selected from the groupconsisting of acrylate, urethane and epoxy.

In certain preferred embodiments, the sample chamber further includes abacking layer that is bond to the bottom layer. The backing layer may bemade of a material selected from the group consisting of polyester,polycarbonate, PMMA, polystyrene and cyclic olefin copolymer.

In yet another aspect, the invention generally relates to a method forfabricating a sample chamber. The method includes: providing anoptically clear backing layer; placing uniformly an irradiation- orthermally-curable layer on the backing layer; imprinting a preformedstructure mold on to the irradiation- or thermally-curable layer,wherein the preformed structured mold protruding into the irradiation-or thermal-curable layer; curing the irradiation- or thermally-curablelayer by irradiation or thermal treatment; removing the preformedstructure mold, thereby forming an imprinted structure in theirradiation- or thermal-cured layer reflecting the preformed structureof the mold; and placing an optically clear top film having an opticallyclear adhesive layer on to the imprinted structure of the irradiation-or thermally-cured layer, thereby forming a chamber having the imprintedstructure.

The method may further include: forming one or more access port on thechamber allowing introduction and removal of a sample.

The optically clear backing layer is made of a material selected fromthe group consisting of polyester, polycarbonate, PMMA, polystyrene andcyclic olefin copolymer.

The irradiation layer may be made of an UV-curable material selectedfrom the group consisting of UV-cured acrylate, urethane and epoxy.

The optically clear top film is made of a material selected from thegroup consisting of polyester, polycarbonate, PMMA, polystyrene andcyclic olefin copolymer.

In some preferred embodiments, the preformed structure mold is formedfrom aluminum mold.

In yet another aspect, the invention generally relates to a method formanufacturing a sample chamber suitable for holding a biological sample.The method includes: imprinting a preformed structure mold on to anirradiation- or thermal-curable layer, the preformed structured moldprotruding into the irradiation- or thermal-curable layer; curing theirradiation- or thermal-curable layer to form an optically clear layerthereby forming a chamber having the imprinted structure; removing thepreformed structure mold; binding an optically clear top film to theimprinted structure of the irradiation- or thermal-cured layer, therebyforming a sample chamber; and forming one or more access port on thechamber allowing introduction and removal of a sample.

Thus, in yet another aspect, the invention generally relates to a methodfor manufacturing a sample chamber suitable for holding a biologicalsample. The method includes: imprinting a preformed structure mold on toa melt plastic layer; cooling the melt plastic layer resulting animprinted structure thereon; removing the preformed structure mold;binding an optically clear top film to the imprinted structure, therebyforming a sample chamber; and forming one or more access port on thechamber allowing introduction and removal of a sample.

In yet another aspect, the invention generally relates to a method formanufacturing a sample chamber suitable for holding a biological sample.The method includes: stamping a hot preformed structure mold on to aplastic layer, resulting an imprinted structure thereon; removing thepreformed structure mold; cooling the plastic layer; binding anoptically clear top film to the imprinted structure, thereby forming asample chamber; and forming one or more access port on the chamberallowing introduction and removal of a sample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a traditional manual bacteria hemacytometer (ColePalmer).

FIG. 2 depicts a schematic cross-section of an exemplary sample chamberaccording to an embodiment of this invention.

FIG. 3 depicts a schematic top view of an exemplary sample chamberaccording to an embodiment of this invention.

FIG. 4 depicts a schematic cross-section of an exemplary sample chamberaccording to an embodiment of this invention.

FIG. 5 depicts a schematic top view of an exemplary sample chamberaccording to an embodiment of this invention.

FIG. 6 is a schematic depiction of a fabrication method.

FIG. 7 depicts an aspect of an exemplary fabrication method according tothe invention.

FIG. 8 depicts an aspect of an exemplary fabrication method according tothe invention.

FIG. 9 depicts an aspect of an exemplary fabrication method according tothe invention.

FIG. 10 depicts an illustration of an exemplary laser distancemeasurement setup and an exemplary image obtained.

FIG. 11 depicts an exemplary thin gap chamber slide according to anembodiment of the invention.

FIG. 12 depicts an exemplary fluorescence of 2.5 μm beads using anexemplary thin gap chamber slide according to an embodiment of theinvention.

FIG. 13 depicts an exemplary fluorescence of 2.5 μm beads using anexemplary thin gap chamber slide according to an embodiment of theinvention.

FIG. 14 depicts an exemplary bright field and fluorescence of 2.5 μmbeads using an exemplary thin gap chamber slide according to anembodiment of the invention.

FIG. 15 depicts an exemplary of bright field image of 2 μm beads usingan exemplary thin gap chamber slide according to an embodiment of theinvention.

FIG. 16 depicts an exemplary of bright field image of yeasts andbacteria using an exemplary thin gap chamber slide according to anembodiment of the invention.

FIG. 17 depicts an exemplary of fluorescence image of yeast and bacteriausing an exemplary thin gap chamber slide according to an embodiment ofthe invention with the signal size presented in a cross section of theimage.

FIG. 18 depicts an exemplary of fluorescence image of yeast and bacteriausing an exemplary thin gap chamber slide according to an embodiment ofthe invention and stained with SYTO9 (Invitrogen) instead of AcridineOrange.

DETAILED DESCRIPTION OF THE INVENTION

The invention is based, in part, on the discovery of unique samplechamber designs and efficient and cost-effective fabrication andmanufacturing methods that allow accurate, consistent and efficientimaging of bacteria or parasites using both bright field andfluorescent.

Bacteria have a wide diversity of shapes and sizes. Bacterial cellstypically are about one tenth the size of eukaryotic cells and aretypically 0.3-5.0 micrometers in length. Among the smallest bacteria aremembers of the genus Mycoplasma, which measure only 0.3 micrometers, assmall as the largest viruses. Some bacteria may be even smaller. Mostbacterial species are either spherical or rod-shaped although othershapes do exist. Many bacterial species exist simply as single cells,others associate in characteristic patterns such as pairs, chains, andclusters. Bacteria can also be elongated to form filaments.

When bacteria form a parasitic association with other organisms, theybecome pathogens. Pathogenic bacteria are a major cause of human deathand disease and cause various infections such as tetanus, typhoid fever,diphtheria, syphilis, cholera, foodborne illness, leprosy andtuberculosis.

Bacteria and parasites have been imaged using manual cell countinghemacytometer, flow cytometer, plate reader, as well as variousfluorescent and electron microscopy and atomic force microscopy. Oneexample is the traditional method of manual cell counting as exemplifiedby the bacteria hemacytometer of Cole Palmer (FIG. 1). It uses capillaryaction to distribute cells into an H-shaped moat that forms two countingareas with enhanced Neubauer rulings. An V-slash at loading sideprovides fluid loading. When viewed through a microscope, hemacytometersdisplay white Neubauer markings against a dark background. Cover slipscover moat region 0.1 mm above ruled surface.

More recently, certain instruments have been developed that are usefulfor imaging and counting cells and biological materials includingcertain bacteria and parasites. An example is an instrument developed byNexcelom Bioscience (Cellometer Vision 10X), which is an automated cellcounting instrument that utilizes a microfluidic counting chamber andcan automatically count cells larger than 2 μm in diameter.

A significant limitation of the existing instruments and methods is thatnone of them provide automatic sample imaging and cell counting that areefficient and provide accurate and consistent results while at the sametime are cost-effective. A major reason for this limitation has been thelack of an enabling sample (or cell) chamber design and correspondingefficient and low cost fabrication and manufacturing methods.

The present invention provides sample chamber designs and fabricationmethods that allow efficient and effective imaging of small sizebiological particles such bacteria or parasites using both bright fieldand fluorescent.

Referring to FIG. 2, which illustrates the cross section of a cellcounting chamber according to an embodiment of this invention. The cellcounting chamber 200 has a bottom layer 210, which may be a layer of aUV-cured material. On the bottom layer 210, certain pre-designed cavity230 is present (profiled on the top surface 215 of the bottom layer210). An optically clear adhesive layer 220 is placed between the bottomlayer 210 and a top layer 240, bonding the top surface 215 of the bottomlayer 210 and the bottom surface 245 of the top layer 240 (with theexception where the pre-designed cavity 230 is present). Additionally,at least one (and preferably two FIG. 3) sample port (e.g., acircular-shaped opening) is present allowing access to the cavity 230.

Referring to FIG. 4, which depicts a cross section of another exemplaryembodiment of the present invention. Here, sample chamber 400 has twosample holding cavities, 432 and 438 with fixed heights h₁ and h₂,respectively. The fixed heights h₁ and h₂ may be identical or different.The corresponding sample ports (FIG. 5) allow introduction of samples tothe two cavities individually, therefore enabling side-by-side imagingof two different samples.

In order to develop a fabrication method that is cost effective andefficient for imaging cytometry, a very important issue has to beaddressed, i.e., the enclosure of the counting chamber. The chamberdepth could be fabricated in various ways, but the most efficient methodas disclosed herein employs imprint lithography. This method utilizes amold that, when imprinted with a thermally or light cured solution,forms a pre-designed structure on a plastic sheet. To avoid expensiveequipments and multiple steps, a unique method as disclosed hereinutilizes an optical clear adhesive to cover the entire top surface thatprovides clear imaging capability while enclosing the chamber.

FIG. 6 depicts an exemplary embodiment of a fabrication method accordingto this invention. A UV-curable solution 610 is dispensed over a plasticsurface 620 and a structure mold 630 is imprinted on the surface 615 ofthe solution 610. UV light is allowed to cure the solution 610. The mold630 is removed and the desired cavity structure 640 is formed. Then, atop layer 650 with an adhesive layer 660 is placed on top of theUV-cured layer 610 with structure 640.

An important aspect of the invention lies in the utilization ofoptically clear transfer adhesive as the enclosing layer of the thin gapchamber, which simplified the method from using an expensive and tediousink transfer system or adhesive coating to simply laying down a topsheet to form the chamber enclosure on the molded structure.

By using a replica molding technique, structures with different gapsizes can be fabricated, which can provide sample concentration-specificchambers. The unique fabrication method can be incorporated into amanufacture regime, such that the slides are made with 2 protectivefilms on top and bottom (FIG. 7). Furthermore, tracking holes can beused to advance sheet processed slides similar to that of the analogcameras with advancing films (FIG. 8). Additionally, the slides can bereplicated on rolls of plastic, which can further reduce the cost andtime of manufacturing (FIG. 9). The counting chambers can bemass-manufactured using rolls of plastic and adhesive, that wouldsignificantly reduce the time and cost of the process.

In one aspect, the invention generally relates to a sample chambersuitable for holding a liquid sample for optical imaging. The samplechamber includes: a top layer; an adhesive layer; a bottom layer; asample holding cavity between the adhesive layer and the bottom layerhaving a fixed depth of from about 0.5 μm to about 1,000 μm; and anassess port for placing a sample into or out of the chamber; wherein atleast a portion of the adhesive layer is optically clear in the range ofabout 250 nm to about 10,000 nm in wavelength, and wherein at least aportion of the top layer and the bottom layer is optically clear in therange of about 300 nm and about 10,000 nm in wavelength.

The bottom layer of the sample chamber further includes a base film.

In certain embodiments, the sample holding cavity of the sample chamberhas a fixed depth of from about 1 μm to about 100 μm. In certainpreferred embodiments, the sample holding cavity has a fixed depth offrom about 2 μm to about 50 μm. In certain preferred embodiments, thesample holding cavity has a fixed depth of from about 2 μm to about 20μm. In certain other preferred embodiments, the sample holding cavityhas a fixed depth less than about 2 μm. In certain other preferredembodiments, the sample holding cavity has a fixed depth less than about1 μm.

In some embodiments, at least a portion of the adhesive layer isoptically clear in the range of about 300 nm to about 10,000 nm inwavelength, and wherein at least a portion of the top and the bottomlayers is optically clear in the range of about 300 nm and about 10,000nm in wavelength.

The top layer of the sample chamber may be made of a material selectedfrom the group consisting of polyester, polycarbonate, poly(methylmethacrylate) (PMMA), polystyrene and cyclic olefin copolymer, and otheroptically clear plastic films.

The adhesive layer of the sample chamber may be a pressure-sensitiveadhesive layer that includes a material selected from acrylic oracrylate adhesive.

The bottom layer may include an irradiation or thermally cured material.For example, the bottom layer may be a UV-cured polymeric material. Incertain preferred embodiments, the UV-cured materials are selected fromthe group consisting of UV-cured acrylate, urethane and epoxy.

In certain preferred embodiments, the sample chamber further includes abacking layer that is bond to the bottom layer. The backing layer mayinclude a material selected from the group consisting of polyester,polycarbonate, PMMA, polystyrene and cyclic olefin copolymer, and otheroptically clear plastic films.

In another aspect, the invention generally relates to a sample chambersuitable for holding a biological sample for optical imaging. The samplechamber includes: an optically clear bottom layer; an optically cleartop layer; an optically clear adhesive layer binding the top layer onone side and forming a sample holding chamber on the other side with anirradiation- or thermally-cured polymer layer, wherein the sampleholding chamber having a fixed depth from the adhesive layer to theirradiation- or thermally-cured polymer layer of from about 0.5 μm toabout 1,000 μm; an inlet port for introducing a sample into the chamber;and an outlet port for removing a sample from the chamber (the sampleholding cavity).

In some embodiments, the sample holding chamber has a fixed depth offrom about 1 μm to about 100 μm. In certain preferred embodiments, thesample holding chamber has a fixed depth of from about 2 μm to about 50μm. In certain preferred embodiments, the sample holding chamber has afixed depth of from about 2 μm to about 20 μm. In certain otherpreferred embodiments, the sample holding chamber has a fixed depth ofless than about 2 μm. In certain other preferred embodiments, the sampleholding chamber has a fixed depth of less than about 1 μm.

For some applications, it is preferred that at least a portion of theadhesive layer is optically clear in the range of about 250 nm to about10,000 nm in wavelength.

The top layer may be made of a material selected from the groupconsisting of polyester, polycarbonate, PMMA, polystyrene and cyclicolefin copolymer.

The adhesive layer may be a pressure-sensitive adhesive layer comprisinga material selected from acrylic or acrylate adhesive.

The bottom layer may include an irradiation or thermally cured material.In certain preferred embodiments, the bottom layer comprises a UV-curedpolymeric material, for example, a material selected from the groupconsisting of acrylate, urethane and epoxy.

In certain preferred embodiments, the sample chamber further includes abacking layer that is bond to the bottom layer. The backing layer may bemade of a material selected from the group consisting of polyester,polycarbonate, PMMA, polystyrene and cyclic olefin copolymer.

In yet another aspect, the invention generally relates to a method forfabricating a sample chamber. The method includes: providing anoptically clear backing layer; placing uniformly an irradiation- orthermally-curable layer on the backing layer; imprinting a preformedstructure mold on to the irradiation- or thermally-curable layer,wherein the preformed structured mold protruding into the irradiation-or thermal-curable layer; curing the irradiation- or thermally-curablelayer by irradiation or thermal treatment; removing the preformedstructure mold, thereby forming an imprinted structure in theirradiation- or thermal-cured layer reflecting the preformed structureof the mold; and placing an optically clear top film having an opticallyclear adhesive layer on to the imprinted structure of the irradiation-or thermally-cured layer, thereby forming a chamber having the imprintedstructure.

The method may further include: forming one or more access port on thechamber allowing introduction and removal of a sample.

The optically clear backing layer is made of a material selected fromthe group consisting of polyester, polycarbonate, PMMA, polystyrene andcyclic olefin copolymer.

The irradiation layer may be made of an UV-curable material selectedfrom the group consisting of UV-cured acrylate, urethane and epoxy.

The optically clear top film is made of a material selected from thegroup consisting of polyester, polycarbonate, PMMA, polystyrene andcyclic olefin copolymer.

In some preferred embodiments, the preformed structure mold is formedfrom aluminum mold.

In some embodiments, an adhesive is coated onto the top layer (e.g.,film) for support, and the top layer and the adhesive layer together aredirectly applied onto the structured surface of the bottom layer to makethe chamber.

In yet another aspect, the invention generally relates to a method formanufacturing a sample chamber suitable for holding a biological sample.The method includes: imprinting a preformed structure mold on to anirradiation- or thermal-curable layer, the preformed structured moldprotruding into the irradiation- or thermal-curable layer; curing theirradiation- or thermal-curable layer to form an optically clear layerthereby forming a chamber having the imprinted structure; removing thepreformed structure mold; binding an optically clear top film to theimprinted structure of the irradiation- or thermal-cured layer, therebyforming a sample chamber; and forming one or more access port on thechamber allowing introduction and removal of a sample.

A desired structure can be formed by thermal casting (e.g., directly onthe bottom layer by casting a melting plastic onto a mold surface toform the bottom part. A desired structure can be formed by thermalstamping (e.g., to thermal stamp a plastic film to form the structure).These processes only involve employ heat to “shape” a plastic materialto form the needed structures on the plastic material and no chemicalreaction is needed. The invention also relates to sample chamberswherein the bottom layer is formed by thermal casting or thermalstamping.

Thus, in yet another aspect, the invention generally relates to a methodfor manufacturing a sample chamber suitable for holding a biologicalsample. The method includes: imprinting a preformed structure mold on toa melt plastic layer; cooling the melt plastic layer resulting animprinted structure thereon; removing the preformed structure mold;binding an optically clear top film to the imprinted structure, therebyforming a sample chamber; and forming one or more access port on thechamber allowing introduction and removal of a sample.

In yet another aspect, the invention generally relates to a method formanufacturing a sample chamber suitable for holding a biological sample.The method includes: stamping a hot preformed structure mold on to aplastic layer, resulting an imprinted structure thereon; removing thepreformed structure mold; cooling the plastic layer; binding anoptically clear top film to the imprinted structure, thereby forming asample chamber; and forming one or more access port on the chamberallowing introduction and removal of a sample.

Samples that may be analyzed using the methods of the invention includebiological materials obtained from or derived from living organisms.Typically the sample will include bacteria, parasites, cells, tissues,or biomolecules, such as proteins, polynucleotides (e.g., DNA or RNA),organic material, and any combination of the foregoing. Such samplesinclude, but are not limited to, hair, skin, tissue, cultured cells,cultured cell media, and body fluids.

A tissue is a mass of connected cells and/or extracellular matrixmaterial, e.g., CNS tissue, neural tissue, eye tissue, liver tissue,placental tissue, mammary gland tissue, gastrointestinal tissue,musculoskeletal tissue, genitourinary tissue, and the like, derivedfrom, for example, a human or other mammal and includes the connectingmaterial and the liquid material in association with the cells and/ortissues. A body fluid is a liquid material derived from, for example, ahuman or other mammal. Such body fluids include, but are not limited to,blood, plasma, serum, serum derivatives, bile, phlegm, saliva, sweat,amniotic fluid, mammary fluid, and cerebrospinal fluid (CSF), such aslumbar or ventricular CSF. A sample also may be media containing cellsor biological material.

Systems of the invention can also be used to interrogate cell lines.Cell lines refer to specific cells that can grow indefinitely given theappropriate medium and conditions. Systems of the invention can be usedto interrogate any type of cell line. Cell lines can be mammalian celllines, insect cell lines or plant cell lines. Exemplary cell lines caninclude tumor cell lines or stem cell lines.

EXAMPLES

An aluminum mold was placed on a 30 mil PC (polycarbonate) sheet withthe green liner removed. Then, a solution of a UV-curable polymer waspipetted on to the surface of the aluminum mold with approximately 5large drops across the entire surface. A 30 mil (a “mil” is 1,000^(th)of an inch) PC sheet (cut into 4 pieces) was then placed on top of thealuminum mold and the UV-curable solution was allowed to spread acrossthe surface. The entire set was cured in a UV chamber for about 12-15seconds. The top 30 mil PC sheet was removed with the structure of thechambers imprinted on the surface. A standard top sheet of a Nexcelomcounting slide (Model No. CHT4-PD, CHT4-SD) was cut out and bottom linerremoved. An optically clear transfer adhesive was placed on the bottomof the top slide sheet. Two holes were punched to form the fluidic inletand outlet ports. The bottom liner of the transfer adhesive was removedand rolled on to the cured imprinted structure with a metal roller toprevent adhesive from touching down to the surface. Finally, the extraplastic area was cut off.

Several parameters were tested. First, the size of the gap in thechamber was measured using a home-built laser position detector whichmeasured the reflection of light from each layer of the slide (see FIG.10). The measured distance was used to calculate the gap size. Secondly,fluorescence from 2.5 μm beads within the chamber was measured to testthe fluorescence capability of the slides and the auto fluorescence fromthe optical adhesive. Thirdly, a well-collimated illuminating lightsource was used in order to prevent a bright center in the image.Finally, fluidic flow characteristics in thin gap chambers were found tomeet the requirements.

The thin gap chamber slide is shown in FIG. 11. The gap size using thelaser position detector measured approximately 30 μm depth. The laserposition detector used a 532 nm green laser that is focused on to thechamber. The reflection of light at different layer produced a brightlight, which was used to estimate the chamber depth. The fluorescence of2.5 μm beads (linearFlow, Invitrogen) in the thin gap chamber wasobserved for both green and orange emission fluorescence channel. FIG.12 shows the bright field (right) and fluorescence image (left) of the2.5 μm beads in a thin gap chamber for green emission. FIG. 13 shows thebright field (right) and fluorescence image (left) of the 2.5 μm beadsin a thin gap chamber for orange emission. FIG. 14 shows the brightfield (right) and fluorescence image (left) of the 2.5 μm beads in athin gap chamber for blue emission.

In another example, 2 μm beads were used. In order to improve the flowof solution into the chamber, 2.5, 5, 10, and 20% IPA (isopropylalcohol) solution was mixed with the 2 μm beads, which enhanced the flowspeed dramatically from greater than 5 min to less than 5 seconds(approximately 4, 3, 2, and 1 second for 2.5, 5, 10, and 20% IPA,respectively). FIG. 15 shows the bright field image of 2 μm beads in afabricated thin gap chamber, to show the dispersion of beads inside thechamber. The beads were observed to continuously flow for a long periodof time before settling down. By taping both outlet and inlet ports withscotch tape, the settle time were reduced from about 15 min to less thanabout 5 min. The temperature gradient from the light did not affect theflow of beads.

Yeast and bacteria samples were imaged (FIGS. 16-18) and the resultsdemonstrate the effectiveness of the cell chamber and methods of theinvention. In FIG. 16, by utilizing 20× objective and thin gap chamber,yeasts and yogurt bacteria were simultaneously observed in the brightfield image. The yeasts and bacteria sample was stained with AcridineOrange (AO), which is a nuclear stain that stains all cells. Thefluorescence image of yeasts and bacteria is shown in FIG. 17, and thesignals are shown in a cross section of the image. Furthermore, otherdyes such as SYTO9 (Invitrogen) may be used to perform the same stainingquality as AO (FIG. 18).

INCORPORATION BY REFERENCE

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made in this disclosure. All such documents arehereby incorporated herein by reference in their entirety for allpurposes.

EQUIVALENTS

The representative examples are intended to help illustrate theinvention, and are not intended to, nor should they be construed to,limit the scope of the invention. Indeed, various modifications of theinvention and many further embodiments thereof, in addition to thoseshown and described herein, will become apparent to those skilled in theart from the full contents of this document, including the examples andthe references to the scientific and patent literature included herein.The examples contain important additional information, exemplificationand guidance which can be adapted to the practice of this invention inits various embodiments and equivalents thereof.

What is claimed is:
 1. A method for fabricating a sample chamber, themethod comprising: providing an optically clear backing layer; placinguniformly an irradiation- or thermally-curable layer on the backinglayer; imprinting a preformed structure mold on to the irradiation- orthermally-curable layer, wherein the preformed structured moldprotruding into the irradiation- or thermal-curable layer; curing theirradiation- or thermally-curable layer by irradiation or thermaltreatment; removing the preformed structure mold, thereby forming animprinted structure in the irradiation- or thermal-cured layerreflecting the preformed structure of the mold; and placing an opticallyclear top film having an optically clear adhesive layer on to theimprinted structure of the irradiation- or thermally-cured layer,thereby forming a chamber having the imprinted structure.
 2. The methodof claim 1, further comprising: forming one or more access port on thechamber allowing introduction and removal of a sample.
 3. The method ofclaim 1, wherein the optically clear backing layer is made of a materialselected from the group consisting of polyester, polycarbonate, PMMA,polystyrene and cyclic olefin copolymer.
 4. The method of claim 1,wherein the irradiation- or thermally-curable layer is made of anirradiation-curable material.
 5. The method of claim 1, wherein theirradiation- or thermally-curable layer is made of an UV-curablematerial selected from the group consisting of UV-cured acrylate,urethane and epoxy.
 6. The method of claim 1, wherein the irradiation-or thermally-curable layer is made of a thermally-curable material. 7.The method of claim 1, wherein the optically clear top film is made of amaterial selected from the group consisting of polyester, polycarbonate,PMMA, polystyrene and cyclic olefin copolymer.
 8. The method of claim 1,wherein the preformed structure mold is formed from aluminum mold.
 9. Amethod for manufacturing a sample chamber suitable for holding abiological sample, the method comprising: imprinting a preformedstructure mold on to an irradiation- or thermal-curable layer, thepreformed structured mold protruding into the irradiation- orthermal-curable layer; curing the irradiation- or thermal-curable layerto form an optically clear layer thereby forming a chamber having theimprinted structure; removing the preformed structure mold; binding anoptically clear top film to the imprinted structure of the irradiation-or thermal-cured layer, thereby forming a sample chamber; and formingone or more access port on the chamber allowing introduction and removalof a sample.
 10. The method of claim 9, wherein the irradiation- orthermal-curable layer is an irradiation-curable layer.
 11. The method ofclaim 10, wherein the irradiation-curable layer is a UV-curable layer.12. The method of claim 11, wherein the UV-curable layer is selectedfrom the group consisting of UV-cured acrylate, urethane and epoxy. 13.The method of claim 9, wherein the optically clear top film is bound tothe imprinted structure via an optically clear adhesive layer.
 14. Themethod of claim 9, wherein the optically clear adhesive layer isselected from the group consisting of polyester, polycarbonate, PMMA,polystyrene and cyclic olefin copolymer.